T-cell-specific deletion of a polypeptide N-acetylgalactosaminyl-transferase gene by site-directed recombination.

UDP-N-acetylgalactosamine (GalNAc): polypeptide N-acetylgalactosaminyltransferase (polypeptide GalNAc-T) catalyzes transfer of the monosaccharide GalNAc to serine and threonine residues, thereby initiating O-linked oligosaccharide biosynthesis. Previous studies have suggested the possibility of multiple polypeptide GalNAc-Ts, although attachment of saccharide units to polypeptide or lipid in generating oligosaccharide structures in vertebrates has been dependent upon the activity of single gene products. To address this issue and to determine the relevance of Oglycosylation variation in T-cell ontogeny, we have directed Cre/loxP mutagenic recombination to the polypeptide GalNAc-T locus in gene-targeted mice. Resulting deletion in the catalytic region of polypeptide GalNAc-T occurred to completion on both alleles in thymocytes and was found in peripheral T cells, but not among other cell types. Thymocyte O-linked oligosaccharide formation persisted in the absence of a functional targeted polypeptide GalNAc-T allele as determined by O-glycan-specific lectin binding. T-cell development and colonization of secondary lymphoid organs were also normal. These results indicate a complexity in vertebrate O-glycan biosynthesis that involves multiple polypeptide GalNAc-Ts. We infer the potential for protein-specific O-glycan formation governed by distinct polypeptide GalNAc-Ts.

The rms1 mutant of pea has elevated indole-3-acetic acid levels and reduced root-sap zeatin riboside content but increased branching controlled by graft-transmissible signal(s)

Rms1 is one of the series of five ramosus loci in pea (Pisum sativum L.) in which recessive mutant alleles confer increased branching at basal and aerial vegetative nodes. Shoots of the nonallelic rms1 and rms2 mutants are phenotypically similar in most respects. However, we found an up to 40-fold difference in root-sap zeatin riboside ([9R]Z) concentration between rms1 and rms2 plants. Compared with wild-type (WT) plants, the concentration of [9R]Z in rms1 root sap was very low and the concentration in rms2 root sap was slightly elevated. To our knowledge, the rms1 mutant is therefore the second ramosus mutant (rms4 being the first) to be characterized with low root-sap [9R]Z content. Like rms2, the apical bud and upper nodes of rms1 plants contain elevated indole-3-acetic acid levels compared with WT shoots. Therefore, the rms1 mutant demonstrates that high shoot auxin levels and low root-sap cytokinin levels are not necessarily correlated with increased apical dominance in pea. A graft-transmissible basis of action has been demonstrated for both mutants from reciprocal grafts between mutant and WT plants. Branching was also largely inhibited in rms1 shoots when grafted to rms2 rootstocks, but was not inhibited in rms2 shoots grafted to rms1 rootstocks. These grafting results are discussed, along with the conclusion that hormone-like signals other than auxin and cytokinin are also involved.

Effects of Cor15a-IPT Gene Expression on Leaf Senescence in Transgenic Petunia x hybrida and Dendranthema x grandiflorum.

To prevent leaf senescence of young transplants or excised shoots during storage under dark and cold conditions, the cytokinin biosynthetic gene isopentenyl transferase (ipt) was placed under the control of a cold-inducible promoter cor15a from Arabidopsis thaliana and introduced into Petunia x hybrida 'Marco Polo Odyssey' and Dendranthema x grandiflorum (chrysanthemum) 'Iridon'. Transgenic cor15a-ipt petunia and chrysanthemum plants and excised leaves remained green and healthy during prolonged dark storage (4 weeks at 25 degrees C) after an initial exposure to a brief cold-induction period (4 degrees C for 72 h). However, cor15a-ipt chrysanthemum plants and excised leaves that were not exposed to a cold-induction period, senesced under the same dark storage conditions. Regardless of cold-induction treatment, leaves and plants of non-transformed plants senesced under prolonged dark storage. Analysis of ipt expression indicated a marked increase in gene expression in intact transgenic plants as well as in isolated transgenic leaves exposed to a short cold-induction treatment prior to dark storage. These changes correlated with elevated concentrations of cytokinins in transgenic leaves after cold treatment. Cor15a-ipt transgenic plants showed a normal phenotype when grown at 25 degrees C.

The gene for albicidin detoxification from Pantoea dispersa encodes an esterase and attenuates pathogenicity of Xanthomonas albilineans to sugarcane

Albicidin phytotoxins are pathogenicity factors in a devastating disease of sugarcane known as leaf scald, caused by Xanthomonas albilineans. A gene (albD) from Pantoea dispersa has been cloned and sequenced and been shown to code for a peptide of 235 amino acids that detoxifies albicidin, The gene shows no significant homology at the DNA or protein level to any known sequence, but the gene product contains a GxSxG motif that is conserved in serine hydrolases, The AlbD protein, purified to homogeneity by means of a glutathione S-transferase gene fusion system, showed strong esterase activity on p-nitrophenyl butyrate and released hydrophilic products during detoxification of albicidins. AlbD hydrolysis of p-nitrophenyl butyrate and detoxification of albicidins required no complex cofactors, Both processes were strongly inhibited by phenylmethylsulfonyl fluoride, a serine enzyme inhibitor, These data strongly suggest that AlbD is an albicidin hydrolase, The enzyme detoxifies albicidins efficiently over a pH range from 5.8 to 8.0, with a broad temperature optimum from 15 to 35 degrees C, Expression of albD in transformed X. albilineans strains abolished the capacity to release albicidin toxins and to incite disease symptoms in sugarcane, The gene is a promising candidate for transfer into sugarcane to confer a form of disease resistance.

Use of the frc gene as a molecular marker to characterize oxalate-oxidizing bacterial abundance and diversity structure in soil.

Oxalate catabolism, which can have both medical and environmental implications, is performed by phylogenetically diverse bacteria. The formyl-CoA-transferase gene was chosen as a molecular marker of the oxalotrophic function. Degenerated primers were deduced from an alignment of frc gene sequences available in databases. The specificity of primers was tested on a variety of frc-containing and frc-lacking bacteria. The frc-primers were then used to develop PCR-DGGE and real-time SybrGreen PCR assays in soils containing various amounts of oxalate. Some PCR products from pure cultures and from soil samples were cloned and sequenced. Data were used to generate a phylogenetic tree showing that environmental PCR products belonged to the target physiological group. The extent of diversity visualised on DGGE pattern was higher for soil samples containing carbonate resulting from oxalate catabolism. Moreover, the amount of frc gene copies in the investigated soils was detected in the range of 1.64x10(7) to 1.75x10(8)/g of dry soil under oxalogenic tree (representing 0.5 to 1.2% of total 16S rRNA gene copies), whereas the number of frc gene copies in the reference soil was 6.4x10(6) (or 0.2% of 16S rRNA gene copies). This indicates that oxalotrophic bacteria are numerous and widespread in soils and that a relationship exists between the presence of the oxalogenic trees Milicia excelsa and Afzelia africana and the relative abundance of oxalotrophic guilds in the total bacterial communities. This is obviously related to the accomplishment of the oxalate-carbonate pathway, which explains the alkalinization and calcium carbonate accumulation occurring below these trees in an otherwise acidic soil. The molecular tools developed in this study will allow in-depth understanding of the functional implication of these bacteria on carbonate accumulation as a way of atmospheric CO(2) sequestration.

The gene for albicidin detoxification from Pantoea dispersa encodes an esterase and attenuates pathogenicity of Xanthomonas albilineans to sugarcane

Albicidin phytotoxins are pathogenicity factors in a devastating disease of sugarcane known as leaf scald, caused by Xanthomonas albilineans. A gene (albD) from Pantoea dispersa has been cloned and sequenced and been shown to code for a peptide of 235 amino acids that detoxifies albicidin. The gene shows no significant homology at the DNA or protein level to any known sequence, but the gene product contains a GxSxG motif that is conserved in serine hydrolases. The AlbD protein, purified to homogeneity by means of a glutathione S-transferase gene fusion system, showed strong esterase activity on p-nitrophenyl butyrate and released hydrophilic products during detoxification of albicidins. AlbD hydrolysis of p-nitrophenyl butyrate and detoxification of albicidins required no complex cofactors. Both processes were strongly inhibited by phenylmethylsulfonyl fluoride, a serine enzyme inhibitor. These data strongly suggest that AlbD is an albicidin hydrolase. The enzyme detoxifies albicidins efficiently over a pH range from 5.8 to 8.0, with a broad temperature optimum from 15 to 35°C. Expression of albD in transformed X. albilineans strains abolished the capacity to release albicidin toxins and to incite disease symptoms in sugarcane. The gene is a promising candidate for transfer into sugarcane to confer a form of disease resistance.

Extratos de Ascophyllum nodosum no tratamento de sementes de milho e soja: avaliações fisiológicas e moleculares

A obtenção de uma lavoura com população adequada de plantas depende da utilização de diferentes práticas agronômicas, estando o sucesso condicionado ao uso de sementes de boa qualidade. No entanto, a semeadura dificilmente é realizada em condições ideais o que resulta em problemas na emergência das plantas. Grande é a procura por alternativas que melhorem a germinação e o desenvolvimento inicial dos cultivos resultando em uniformidade de emergência, garantindo o estande e culminando em produção. O tratamento de sementes com agroquímicos fitossanitários é uma solução parcial no combate de agentes fitopatológicos e pragas, mas poucas são as soluções adotadas para melhorar o desenvolvimento vegetal inicial, que pode levar a desuniformidade e falhas no estande gerando prejuízos econômicos. Os extratos de algas já demonstraram em diversos estudos sua eficiência no desenvolvimento vegetal quando aplicados em plantas. Porém poucos são os estudos voltados para os efeitos dos extratos de algas na germinação e emergência. Assim, o intuito deste trabalho foi testar o extrato comercial de Ascophyllum nodosum, e diferentes fracionamentos do mesmo, no tratamento de sementes de soja e milho. Avaliou-se o efeito de diferentes doses no desenvolvimento das plântulas e as doses de melhor resposta foram utilizadas no tratamento de sementes de soja a fim de associar as respostas obtidas à expressão gênica de 9 genes relacionados ao processo germinativo em 24 e 48h de embebição. Sementes de soja tratadas com o extrato comercial resultaram em plântulas menos desenvolvidas o que pode estar relacionado ao alto teor de sais contidos no produto. O tratamento com as demais frações favoreceu o desenvolvimento das plântulas, principalmente o desenvolvimento radicular. Sementes de milho tratadas não apresentaram desenvolvimento tão satisfatório quanto as sementes de soja tratadas. A análise da expressão gênica relativa demonstrou que o tratamento com frações do extrato comercial é capaz de regular algumas vias do metabolismo hormonal, como a isopentenil transferase e a GA20 oxidase 2, e do catabolismo de reservas, como a acil-CoA oxidase. Em condições ótimas, o tratamento de sementes de soja com frações do extrato comercial de A. nodosum favoreceu o desenvolvimento inicial das plântulas de soja, no entanto não ocasionou grandes alterações no desenvolvimento de milho. Este estudo demonstrou a possibilidade de utilização de frações do extrato de A. nodosum no favorecimento do desenvolvimento inicial de plântulas de soja. Maiores estudos são necessários quanto às respostas em campo e na atenuação de estresses para viabilizar seu uso como um bioestimulante em sementes.

High affinity binding of albicidin phytotoxins by the AlbA protein from Klebsiella oxytoca

Albicidins are a family of phytotoxins and antibiotics which play an important role in the pathogenesis of sugarcane leaf scald disease. The albA gene from Klebsiella oxytoca encodes a protein which inactivates albicidin by heat-reversible binding. Albicidin ligand binding to a recombinant AlbA protein, purified by means of a glutathione S-transferase gene fusion system, is an almost instant and saturable reaction. Kinetic and stoichiometric analysis of the binding reaction indicated the presence of a single high affinity binding site with a dissociation constant of 6.4 x 10(-8) M. The AlbA-albicidin complex is stable from 4 to 40 degrees C, from ph 5 to 9 and in high salt solutions. Treatment with protein denaturants released all bound albicidin. These properties indicate that AlbA may be a useful affinity matrix for selective purification of albicidin antibiotics. AlbA does not bind to p-nitrophenyl butyrate or alpha-naphthyl butyrate, the substrates of the albicidin detoxification enzyme AlbD from Pantoea dispersa. The potential exists to pyramid genes for different mechanisms in transgenic plants to protect plastid DNA replication from inhibition by albicidins.

Secretory pathway of trypanosomatid parasites

The Trypanosomatidae comprise a large group of parasitic protozoa, some of which cause important diseases in humans. These include Tryanosoma brucei (the causative agent of African sleeping sickness and nagana in cattle), Trypanosoma cruzi (the causative agent of Chagas' disease in Central and South America), and Leishmania spp. (the causative agent of visceral and [muco]cutaneous leishmaniasis throughout the tropics and subtropics). The cell surfaces of these parasites are covered in complex protein- or carbohydrate-rich coats that are required for parasite survival and infectivity in their respective insect vectors and mammalian hosts. These molecules are assembled in the secretory pathway. Recent advances in the genetic manipulation of these parasites as well as progress with the parasite genome projects has greatly advanced our understanding of processes that underlie secretory transport in trypanosomatids. This article provides an overview of the organization of the trypanosomatid secretory pathway and connections that exist with endocytic organelles and multiple lytic and storage vacuoles. A number of the molecular components that are required for vesicular transport have been identified, as have some of the sorting signals that direct proteins to the cell surface or organelles it? the endosome-vacuole system. Finally, the subcellular organization of the major glycosylation pathways in these parasites is reviewed. Studies on these highly divergent eukaryotes provide important insights into the molecular processes underlying secretory transport that arose very early in eukaryotic evolution. They also reveal unusual or novel aspects of secretory), transport and protein glycosylation that may be exploited in developing new antiparasite drugs.

Effects of radiotherapy with concomitant and adjuvant temozolomide versus radiotherapy alone on survival in glioblastoma in a randomised phase III study: 5-year analysis of the EORTC-NCIC trial.

BACKGROUND: In 2004, a randomised phase III trial by the European Organisation for Research and Treatment of Cancer (EORTC) and National Cancer Institute of Canada Clinical Trials Group (NCIC) reported improved median and 2-year survival for patients with glioblastoma treated with concomitant and adjuvant temozolomide and radiotherapy. We report the final results with a median follow-up of more than 5 years. METHODS: Adult patients with newly diagnosed glioblastoma were randomly assigned to receive either standard radiotherapy or identical radiotherapy with concomitant temozolomide followed by up to six cycles of adjuvant temozolomide. The methylation status of the methyl-guanine methyl transferase gene, MGMT, was determined retrospectively from the tumour tissue of 206 patients. The primary endpoint was overall survival. Analyses were by intention to treat. This trial is registered with Clinicaltrials.gov, number NCT00006353. FINDINGS: Between Aug 17, 2000, and March 22, 2002, 573 patients were assigned to treatment. 278 (97%) of 286 patients in the radiotherapy alone group and 254 (89%) of 287 in the combined-treatment group died during 5 years of follow-up. Overall survival was 27.2% (95% CI 22.2-32.5) at 2 years, 16.0% (12.0-20.6) at 3 years, 12.1% (8.5-16.4) at 4 years, and 9.8% (6.4-14.0) at 5 years with temozolomide, versus 10.9% (7.6-14.8), 4.4% (2.4-7.2), 3.0% (1.4-5.7), and 1.9% (0.6-4.4) with radiotherapy alone (hazard ratio 0.6, 95% CI 0.5-0.7; p<0.0001). A benefit of combined therapy was recorded in all clinical prognostic subgroups, including patients aged 60-70 years. Methylation of the MGMT promoter was the strongest predictor for outcome and benefit from temozolomide chemotherapy. INTERPRETATION: Benefits of adjuvant temozolomide with radiotherapy lasted throughout 5 years of follow-up. A few patients in favourable prognostic categories survive longer than 5 years. MGMT methylation status identifies patients most likely to benefit from the addition of temozolomide. FUNDING: EORTC, NCIC, Nélia and Amadeo Barletta Foundation, Schering-Plough.

Detection of differentially expressed genes in synovial fibroblasts by restriction fragment differential display

Objective. To identify differentially expressed genes in synovial fibroblasts and examine the effect on gene expression of exposure to TNF-alpha and IL-1beta. Methods. Restriction fragment differential display was used to isolate genes using degenerate primers complementary to the lysophosphatidic acid acyl transferase gene family. Differential gene expression was confirmed by reverse transcription-polymerase chain reaction and immunohistochemistry using a variety of synovial fibroblasts, including cells from patients with osteoarthritis and self-limiting parvovirus arthritis. Results. Irrespective of disease process, synovial fibroblasts constitutively produced higher levels of IL-6 and monocyte chemoattractant protein 1 (MCP-1) (CCL2) than skin fibroblasts. Seven genes were differentially expressed in synovial fibroblasts compared with skin fibroblasts. Of these genes, four [tissue factor pathway inhibitor 2 (TFPI2), growth regulatory oncogene beta (GRObeta), manganese superoxide dismutase (MnSOD) and granulocyte chemotactic protein 2 (GCP-2)] were all found to be constitutively overexpressed in synoviocytes derived from patients with osteoarthritis. These four genes were only weakly expressed in other synovial fibroblasts (rheumatoid and self-limiting parvovirus infection). However, expression in all types of fibroblasts was increased after stimulation with TNF-alpha and IL-1beta. Three other genes (aggrecan, biglycan and caldesmon) were expressed at higher levels in all types of synovial fibroblasts compared with skin fibroblasts even after stimulation with TNF-alpha and IL-1. Conclusions. Seven genes have been identified with differential expression patterns in terms of disease process (osteoarthritis vs rheumatoid arthritis), state of activation (resting vs cytokine activation) and anatomical location (synovium vs skin). Four of these genes, TFPI2, GRObeta (CXCL2), MnSOD and GCP-2 (CXCL6), were selectively overexpressed in osteoarthritis fibroblasts rather than rheumatoid fibroblasts. While these differences may represent differential behaviour of synovial fibroblasts in in vitro culture, these observations suggest that TFPI2, GRObeta (CXCL2), MnSOD and GCP-2 (CXCL6) may represent new targets for treatments specifically tailored to osteoarthritis.

Regulation of the gene encoding glutathione S-transferase M1 (GSTM1) by the Myb oncoprotein

The identification of Myb 'target' genes will not only aid in the understanding of how overexpression of Myb, or expression of activated forms of Myb, leads to cellular transformation but will also shed light on its role in normal cells. Using a combination of an estrogen-regulated Myb-transformed cell line (ERMYB) and PCR-based subtractive hybridization, we have identified the gene (GSTM1) encoding the detoxification enzyme glutathione S-transferase M1 as being transcriptionally upregulated by Myb. Functional analysis of the GSTM1 promoter using reporter assays indicated that both the DNA binding and transactivation domains of Myb were required for transcriptional activation. Mutational analysis of consensus Myb-binding sites (MBS) in the promoter and electrophoretic mobility gel shift analysis indicated that one of the three potential MBS can bind Myb protein, and is the primary site involved in the regulation of this promoter by Myb.

Farnesoic acid O-methyl transferase (FAMeT) isoforms: Conserved traits and gene expression patterns related to caste differentiation in the stingless bee, Melipona scutellaris

Farnesoic acid O-methyl transferase (FAMeT) is the enzyme that catalyzes the formation of methyl farnesoate (MF) from farnesoic acid (FA) in the biosynthetic pathway of juvenile hormone (JH). This work reports the cloning, sequencing, and expression of FAMeT gene from the stingless bee Melipona scutellaris (MsFAMeT). The MsFAMeT in silica analysis showed that greatest sequence similarity is found in Apis mellifera and other insects, while relatively less similarity is shown in crustaceans. Evidence of alternative splicing of a 27 nucleotide (nt) microexon explains the presence of the detected isoforms, 1 and 2. The expression analysis of the two isoforms showed a marked difference when castes were compared, suggesting that they could be involved differently in the JH metabolism in M. scutellaris, providing new insights for the comprehension of female plasticity.

Caracterização funcional do gene da Glutationa-S-Transferase Epsilon 2 (GSTE2) em Aedes aegypti

As Glutationa-S-Transferases (GSTs) em insetos desempenham um papel fundamental na metabolização de inseticidas químicos, e provavelmente estão envolvidas na proteção contra o estresse oxidativo decorrente da exposição a xenobióticos. O objetivo do trabalho foi a caracterização funcional do gene GSTE2 em linhagens de Aedes aegypti com diferentes perfis de susceptibilidade ao temephos. Foram usadas uma colônia susceptível (RecLab) e outra resistente, (RecR). Larvas de ambas as linhagens foram divididas em dois grupos: exposto ao temephos com concentrações subletais e não exposto. Os indivíduos sobreviventes foram usados em ensaios enzimáticos para medir a atividade das GSTs totais contra os substratos CDNB (padrão) e o 4-HNE, um produto endógeno resultante da peroxidação de lipídeos. Adicionalmente, foi feito o sequenciamento do cDNA deste gene em amostras das duas linhagens e a sua expressão foi investigada. A GSTE2 das duas linhagens foi expressa em sistema heterólogo e purificada para avaliação da atividade metabólica contra o 4-HNE, através de testes de biocatálise. Os resultados revelaram que a atividade enzimática da GST usando o CDNB foi normal para RecLab, em ambas as condições estudadas, porém, para RecR houve alteração na atividade de GST, para os dois grugo estudados . Usando o 4-HNE como substrato, as duas linhagens apresentaram um perfil enzimático alterado para GST em relação à Rock, com uma resposta aumentada após a exposição ao temephos. Foram identificados polimorfismos que diferenciam as duas linhagens. Os resultados de expressão gênica indicaram que as larvas resistentes apresentam níveis de expressão significativamente maiores do que as susceptíveis, e em RecR a expressão caiu após a exposição, sugerindo o envolvimento dessa enzima nos processos de resistência metabólica na linhagem RecR. Esses dados abrem novas perspectivas de monitoramento da resistência metabólica em Ae. aegypti